280 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS IRed Lake C Alaine1.00g I IWeigh exactly) •Dissolve with 2% aq. NaOH. 60ml on water bath Transfer the solution into 100ml separating funnel I Extract with 25ml chloroform (Repeat three times) I ! I IAqueous layer I IChloroform layer I I Dehydration through sodium sulfate anhydrous column • Add 1.00ml n-tetradeca Concentrate with rotary evaporator I IHP'C-vl Column: Nucleosil-50, 4mm i.d.x 25cm, Temp.: R.T. Eluent : 20% ethyl acetate/n-hexane, 2.0ml/min. Detector: (i)UV (254nm), 0.02 AUFS (•)FL (Ex----300nm. Eu----330nm), Sen.----3 Inj. vol. : 25/zl Figure 6. Flow chart for determination of aromatic amines in Red Lake C Amine. RELATIONSHIP BETWEEN AROMATIC AMINES IN RED LAKE C AMINE AND SUBSIDIARY COLORS IN D&C RED NO. 9 In order to understand the quantitative relation between aromatic amines in Red Lake C Amine and the subsidiary colors in D&C Red No. 9 and to determine the purity of Red Lake C Amine, D&C Red No. 9s were synthesized with purified Red Lake C Amine and with Red Lake C Amine to which 1, 5, 10, and 50 ppm of three kinds of Table IV Determination of Aromatic Amines in Commercial Red Lake C Amines (ppm) Samples P- 0A P-12A P-15A Manufacturer D 0.13 9.3 7.1 Manufacturer D 0.15 2.8 5.4 Manufacturer D 0.37 6.9 7.0 , Manufacturer E 0.15 15.4 102 Manufacturer F 0.19 9.5 15.3 Manufacturer G 0.27 1.6 1.2 P-OA' Aniline P-12A: 3-Chloro-p-toluidine P-15A: 4-Chloro-m-toluidine

D&C RED NO. 9 ANALYSIS AND PURIFICATION 281 aromatic amines were added. The subsidiary colors produced in the synthesized D&C Red No. 9s were quantitatively analyzed according to the procedure shown in Figure 4. The liquid chromatograms of these D&C Red No. 9s are shown in Figure 7. No I Amount of each of the three aromatic amines' added to the purified Red Lake C Amine 9) ppm & . , 10 ppm I 1 ppm none L, Retention time (minutes) Figure 7. Liquid chromatograms of three subsidiary colors (P-0, P-•[•, and P-15) in synthesized D&C Red No. 9. Column: Partisil*, 6 mm i.d. x 25 cm, Temp.: R.T. Eluent: 4% dichloromethane/n-hexane, 3 ml/min. Detector: VIS (436 nm), 0.02 AUFS *Whatman (USA) subsidiary colors were produced in D&C Red No. 9 synthesized from purified Red Lake C Amine free of aromatic amines. It was found that derivative P-15 of D&C Red No. 9 was not produced in the regular synthetic process by desulfonation, and that the subsidiary colors were produced in proportion to the amounts of added corresponding aromatic amines. The yield of subsidiary colors was approximately 80% in each case. It is estimated from these results that the other subsidiary colors in D&C Red No. 9 are also produced in proportion to the amounts of other aromatic amines contained in Red Lake C Amine. In order to produce D&C Red No. 9 free from subsidiary colors, therefore, it is proposed that Red Lake C Amine free from aromatic amines must be used.